We have analyzed a variety of molecules obtained from various sources to get the quantitative information. Additionally, we are developing methods to improve the quantitative information that can be gained. 1. Method Development. The use of label-free and labeling approaches for quantitative proteomics studies has been implemented. These methodologies involve both data dependent (DDA) and data-independent analyses (Mse). Additionally, we are incorporating ion mobility methods in an effort to gain further information. Ion mobility is a useful tool to aid in mass spectrometry applications because it allows for the measurement of the collisional cross section of a molecule and gives information about the three-dimensional shape of a compound in the gas phase. Ion mobility separates ions based on their differential mobility through a buffer gas based on the ions shape, charge, and mass. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions and thus travel more slowly than those ions that comprise a smaller cross-section. Thus, ion mobility serves as a useful means of orthogonal separation in unrelated molecules as well. This is a real benefit because the ability to identify and quantify proteins is directly linked to the power of chromatographic separations. 2. Nucleoside Study. DNA was isolated from different yeast strains, digested, and analyzed by MS. We performed the MS analyses to determine the levels of dNTPs and NTPs (ribos) that could be detected. Ultimately, nuclear DNA samples were treated with Dnase and ribo identifications were performed to see how much nucleoside is incorporated into DNA. 3. Buprenorphine Study. Buprenorphine is an opiate which is used to control pain in experimental animals by our CMB. Plasma concentrations of buprenorphine drop below therapeutic levels between 2 and 4 hours after subcutaneous injection. To achieve a longer therapeutic window, sustained release formulations and pluronic gel formulations are being investigated. Buprenorphine, norbuprenorphine, and their glucuronides in plasma have been quantified from initial formulations. 4. Eicosanoid Studies. Eicosanoids and related fatty acid metabolites serve as signaling molecules and are intricately involved in inflammation and cardiovascular health. The level of eicosanoids and eicosanoid metabolites are thought to be involved in many diseases. We are involved in a variety of projects measuring these compounds using mass spectrometry. We use liquid chromatography tandem mass spectrometry to analyze a panel of 71 of these molecules which has allowed us to collaborate with several intramural and extramural researchers. We are also developing an untargeted approach to analyze these molecules on another instrument. 5. Steroid Studies. Steroid hormones are widely distributed in nature and are potent signaling molecules. As such, they are of interest to several researchers within the institute. Steroids are often present at low concentrations and exhibit low response in electrospray ionization. We are evaluating chemical derivatization procedures that will increase the signal intensity across the spectrum of hydroxy steroids and keto steroids. 6. BALF Studies. This project involves the pulmonary function of a knockout model of the Low Density Lipoprotein Receptor-related protein 1(LRP1). A novel pulmonary phenotype was identified and the disrupted mechanisms leading to this phenotype are being investigated. The receptor has over 30 ligands making the task of identifying changes in the concentration of any one ligand more difficult. We are using mass spectrometry to assay all of the relative concentrations of the ligands within the bronchoalveolar lavage fluid at one time, in addition to potential targets previously not considered. The data have been collected and the biostatisticians are doing statistics on the results. 7. NAD Studies. Per a request from the laboratory of Dr. Xiaoling Li, the MSRSG has recently developed an LC-MS-based panel for the relative quantitation of metabolites in the NAD pathway. Thus far, the method has been developed to include 11 compounds in the pathway and work is on-going to add 4 other molecules. This panel has been optimized thus far for the analyses of samples arising from cell culture, media, and tissue. In the future we plan to expand these analyses to serum and other biofluids. Currently the MSRSG is retooling this method for absolute quantitation. 8. Comparison Proteomics Study. In collaboration with the Doetsch lab, we are comparing the proteins expressed in HBEC lung epithelial cells and MCF10A breast epithelial cells before and after doxycycline induction. 9. UDP-Hexose. In collaboration with the laboratory of Dr. Don Cook, the MSRSG developed a method for the relative quantitation of UDP-hexose from bronchoalveolar lavage fluid. The Cook laboratory hypothesizes that this molecule becomes upregulated in the lungs upon allergic sensitization.